The stomatal response to CO2 is linked to changes in guard cell zeaxanthin*

The mechanisms mediating CO₂ sensing and light–CO₂ interactions in guard cells are unknown. In growth chamber-grown Vicia faba leaves kept under constant light (500 μ mol m^–2 s^–1) and temperature, guard cell zeaxanthin content tracked ambient [CO₂] and stomatal apertures. Increases in [CO₂] from 400 to 1200 cm³ m^–3 decreased zeaxanthin content from 180 to 80 mmol mol^–1 Chl and decreased stomatal apertures by 7·0 μ m. Changes in zeaxanthin and aperture were reversed when [CO₂] was lowered. Guard cell zeaxanthin content was linearly correlated with stomatal apertures. In the dark, the CO₂-induced changes in stomatal aperture were much smaller, and guard cell zeaxanthin content did not change with chamber [CO₂]. Guard cell zeaxanthin also tracked [CO₂] and stomatal aperture in illuminated stomata from epidermal peels. Dithiothreitol (DTT), an inhibitor of zeaxanthin formation, eliminated CO₂-induced zeaxanthin changes in guard cells from illuminated epidermal peels and reduced the stomatal CO₂ response to the level observed in the dark. These data suggest that CO₂-dependent changes in the zeaxanthin content of guard cells could modulate CO₂-dependent changes of stomatal apertures in the light while a zeaxanthin-independent CO₂ sensing mechanism would modulate the CO₂ response in the dark.     

Silencing of NtMPK4 impairs CO-induced stomatal closure, activation of anion channels and cytosolic Casignals in Nicotiana tabacum guard cells

Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of Nt MPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of Nt MPK4-silenced plants do not close in elevated atmospheric CO₂, and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO₂ activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in Nt MPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca²⁺ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca²⁺ than in those with steady Ca²⁺ levels. Both the darkness-induced anion channel activation and Ca²⁺ signals were repressed in Nt MPK4-silenced guard cells. These data show that CO₂ and darkness can activate anion channels in a Ca²⁺-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca²⁺ concentration. Nt MPK4 plays an essential role in CO₂- and darkness-induced activation of guard-cell anion channels, through Ca²⁺-independent as well as Ca²⁺-dependent signalling pathways.     

Effect of sink size on photosynthesis and carbohydrate content of leaves of three spring wheat varieties

Effects of CO₂ on stomatal movements of Commelina communis L. were studied with plants, epidermal strips and guard cell protoplasts. With plants, the stomatal response induced by a blue light pulse was studied for different ambient CO₂ concentration ranging from CO₂-deprived air to 100 Pa in darkness or under red light. It was observed that the blue light response could be obtained not only under a red light background but also in darkness and CO₂-free air, the two responses being quite similar.
With epidermal strips, the effect of CO₂ on ferricyanide reductase activity at the guard cell plasmalemma was studied by transmission electron microscopy. In the presence of ferric ions, reduced ferricyanide gives an electron dense precipitate of Prussian Blue. In darkness and air, no precipitate was observed. In darkness and CO₂-free air as well as under light and normal air, a precipitate was found along the plasmalemma of the guard cells, indicating a ferricyanide reductase activity. With guard cell protoplasts suspended in a medium either in equilibrium with air or in a CO₂-free medium the H⁺ extrusion induced by a blue light pulse added to a red light background was measured. A low CO₂ content was obtained by adding photosynthetic algae to the suspension of guard cell protoplasts. In a CO₂-free medium the rate of H⁺ extrusion was enhanced.
The results are discussed on the basis of a possible competition for reducing power between CO₂ fixation and a putative blue light dependent redox chain located on the plasma membrane.     

Photosynthesis affects following night leaf conductance in Vicia faba

Night-time stomatal opening in C₃ plants may result in significant water loss when no carbon gain is possible. The objective of this study was to determine if endogenous patterns of night-time stomatal opening, as reflected in leaf conductance, in Vicia faba are affected by photosynthetic conditions the previous day. Reducing photosynthesis with low light or low CO₂ resulted in reduced night-time stomatal opening the following night, irrespective of the effects on daytime stomatal conductance. Likewise, increasing photosynthesis with enriched CO₂ levels resulted in increased night-time stomatal opening the following night. Reduced night-time stomatal opening was not the result of an inability to regulate stomatal aperture as leaves with reduced night-time stomatal opening were capable of greater night-time opening when exposed to low CO₂. After acclimating plants to long or short days, it was found that night-time leaf conductance was greater in plants acclimated to short days, and associated with greater leaf starch and nitrate accumulation, both of which may affect night-time guard cell osmotic potential. Direct measurement of guard cell contents during endogenous night-time stomatal opening will help identify the mechanism of the effect of daytime photosynthesis on subsequent night-time stomatal regulation.     

Stomatal responses to light and CO2 are dependent on KCI concentration

Abstract. The responses of stomata on detached epidermis of Commelina communis to light and CO₂ have been shown to be strongly dependent on the concentration of KCI in the incubation medium. There was a high sensitivity to the two stimuli in 50 mM KCI, but there were much reduced responses at lower and higher concentrations. It is considered that an appropriate choice of medium is essential if useful physiological studies of stomata are to be made using epidermal strips. At lower KCI concentrations, the ability of the stomata to open is thought to be limited by the availability of K⁺ ions, and at higher concentrations their ability to close may be affected because of an inhibition of the net efflux of K⁺. The production of malate was related to KCI concentration, and was largest in the medium containing zero KCI which supported poor stomatal responses to light and CO₂It is concluded that malate metabolism is unlikely to play a central part in the changes in guard cell turgor that are brought about by light and CO₂.     

Carbon dioxide induces increases in guard cell cytosolic free calcium

The hypothesis that increases in cytosolic free calcium ([Ca²⁺]_i) are a component of the CO₂ signal transduction pathway in stomatal guard cells of Commelina communis has been investigated. This hypothesis was tested using fura-2 fluorescence ratio photometry to measure changes in guard cell [Ca²⁺]_i in response to challenge with 700 µl l⁻¹ CO₂. Elevated CO₂ induced increases in guard cell [Ca²⁺]_i which were similar to those previously reported in response to abscisic acid. [Ca²⁺]_i returned to resting values following removal of the CO₂ and further application of CO₂ resulted in a second increase in [Ca²⁺]_i. This demonstrated that the CO₂-induced increases in [Ca²⁺]_i were stimulus dependent. Removal of extracellular calcium both prevented the CO₂-induced increase in [Ca²⁺]_i and inhibited the associated reduction in stomatal aperture. These data suggest that Ca²⁺ acts as a second messenger in the CO₂ signal transduction pathway and that an increase in [Ca²⁺]_i may be a requirement for the stomatal response to CO₂.     

Interaction of elevated CO2 and O3 on growth, photosynthesis and respiration of three perennial species grown in low and high nitrogen

Seedlings of three species native to central North America, a C₃ tree, Populus tremuloides Michx., a C₃ grass, Agropyron smithii Rybd., and a C₄ grass, Bouteloua curtipendula Michx., were grown in all eight combinations of two levels each of CO₂, O₃ and nitrogen (N) for 58 days in a controlled environment. Treatment levels consisted of 360 or 674 μmol mol^-1 CO₂, 3 or 92 nmol mol^-1 O₃, and 0.5 or 6.0 m M N. In situ photosynthesis and relative growth rate (RGR) and its determinants were obtained at each of three sequential harvests, and leaf dark respiration was measured at the second and third harvests. In all three species, plants grown in high N had significantly greater whole-plant mass, RGR and photosynthesis than plants grown in low N. Within a N treatment, elevated CO₂ did not significantly enhance any of these parameters nor did it affect leaf respiration. However, plants of all three species grown in elevated CO₂ had lower stomatal conductance compared to ambient CO₂-exposed plants. Seedlings of P. tremuloides (in both N treatments) and B. curtipendula (in high N) had significant ozone-induced reductions in whole-plant mass and RGR in ambient but not under elevated CO_2. This negative O₃ impact on RGR in ambient CO₂ was related to increased leaf dark respiration, decreased photosynthesis and/or decreased leaf area ratio, none of which were noted in high O₃ treatments in the elevated CO₂ environment. In contrast, A. smithii was marginally negatively affected by high O_3.     

Elevated CO2 enhances stomatal responses to osmotic stress and abscisic acid in Arabidopsis thaliana

A , carbon assimilation rate
ABA, abscisic acid
Ci , intercellular space CO₂ concentration
g , leaf conductance
WUE, water use efficiency

Carbon dioxide and abscisic acid (ABA) are two major signals triggering stomatal closure. Their putative interaction in stomatal regulation was investigated in well-watered air-grown or double CO₂-grown Arabidopsis thaliana plants, using gas exchange and epidermal strip experiments. With plants grown in normal air, a doubling of the CO₂ concentration resulted in a rapid and transient drop in leaf conductance followed by recovery to the pre-treatment level after about two photoperiods. Despite the fact that plants placed in air or in double CO₂ for 2 d exhibited similar levels of leaf conductance, their stomatal responses to an osmotic stress (0·16–0·24 MPa) were different. The decrease in leaf conductance in response to the osmotic stress was strongly enhanced at elevated CO₂. Similarly, the drop in leaf conductance triggered by 1 μ M ABA applied at the root level was stronger at double CO₂. Identical experiments were performed with plants fully grown at double CO₂. Levels of leaf conductance and carbon assimilation rate measured at double CO₂ were similar for air-grown and elevated CO₂-grown plants. An enhanced response to ABA was still observed at high CO₂ in pre-conditioned plants. It is concluded that: (i) in the absence of stress, elevated CO₂ slightly affects leaf conductance in A. thaliana ; (ii) there is a strong interaction in stomatal responses to CO₂ and ABA which is not modified by growth at elevated CO₂.     

Water vapour fluxes and their impact under elevated CO2 in a C4-tallgrass prairie

We measured leaf-level stomatal conductance, xylem pressure potential, and stomate number and size as well as whole plant sap flow and canopy-level water vapour fluxes in a C4-tallgrass prairie in Kansas exposed to ambient and elevated CO₂. Stomatal conductance was reduced by as much as 50% under elevated CO₂ compared to ambient. In addition, there was a reduction in stomate number of the C4 grass, Andropogon gerardii Vitman, and the C3 dicot herb, Salvia pitcheri Torr., under elevated CO₂ compared to ambient. The result was an improved water status for plants exposed to elevated CO₂ which was reflected by a less negative xylem pressure potential compared to plants exposed to ambient CO₂. Sap flow rates were 20 to 30% lower for plants exposed to elevated CO₂ than for those exposed to ambient CO₂. At the canopy level, evapotranspiration was reduced by 22% under elevated CO₂. The reduced water use by the plant canopy under elevated CO₂ extended the photosynthetically-active period when water became limiting in the ecosystem. The result was an increased above- and belowground biomass production in years when water stress was frequent.     

Effect of elevated CO2 on the stomatal distribution and leaf physiology of Alnus glutinosa

Variation in stomatal development and physiology of mature leaves from Alnus glutinosa plants grown under reference (current ambient, 360 μmol mol⁻¹ CO₂) and double ambient (720 μmol mol⁻¹ CO₂) carbon dioxide (CO₂) mole fractions is assessed in terms of relative plant growth, stomatal characters (i.e. stomatal index and density) and leaf photosynthetic characters. This is the first study to consider the effects of elevated CO₂ concentration on the distribution of stomata and epidermal cells across the whole leaf and to try to ascertain the cause of intraleaf variation. In general, a doubling of the atmospheric CO₂ concentration enhanced plant growth and significantly increased stomatal index. However, there was no significant change in relative stomatal density. Under elevated CO₂ concentration there was a significant decrease in stomatal conductance and an increase in assimilation rate. However, no significant differences were found for the maximum rate of carboxylation ( V _cmax) and the light saturated rate of electron transport ( J _max) between the control and elevated CO₂ treatment.     

Physiological effects of long-term exposure to low and moderate concentrations of atmospheric NH3 on poplar leaves

Abstract. Poplar shoots ( Populus euramericana L.) obtained from cuttings were exposed for 6 or 8 weeks to NH₃ concentrations of 50 and 100 μgm⁻³ or filtered air in fumigation chambers. After this exposure the rates of NH₃ uptake, transpiration, CO₂ assimilation and respiration of leaves were measured using a leaf chamber. During the long-term exposure also modulated chlorophyll fluorescence measurements were carried out to obtain information about the photosynthetic performance of individual leaves. Both fluorescence and leaf chamber measurements showed a higher photosynthetic activity of leaves exposed to 100 μg NH₃ m⁻³. These leaves showed also a larger leaf conductance and a larger uptake rate of NH₃ than leaves exposed to 50 μg m⁻³ NH₃ or filtered air. The long-term NH₃ exposure did not induce an internal resistance against NH₃ transport in the leaf, nor did it affect the leaf cuticle. So, not only at a short time exposure, but also at a long-term exposure NH₃ uptake into leaves can be calculated from data on the boundary layer and stomatal resistance for H₂O and ambient NH₃-concentration. Furthermore, the NH₃ exposure had no effect on the relation between CO₂-assimilation and stomatal conductance, indicating that NH₃ in concentrations up to 100 μg m⁻³ has no direct effect on stomatal behaviour; for example, by affecting the guard or contiguous cells of the stomata.     

Evolutionary responses of stomatal density to global CO2 change

Stomatal density is known to respond to CO₂ levels during leaf development. Current interest in the increasing concentration of atmospheric CO₂ has stimulated much experimentation on the responses of plants to relatively short-term exposure in artificially high CO₂ levels. Attempts to extrapolate from short-term to long-term responses raise fundamental questions concerning evolutionary change in response to rising global CO₂ levels. We consider the improved water use efficiency observed under elevated CO₂ levels to be the main driving force of natural selection affecting the genotypic component controlling stomatal density. Whether a response is merely phenotypic or becomes incorporated into the genotype depends on two factors: (i) the time scale of exposure and (ii) the generation time of a species. Measurements of stomatal density on fossil leaves of Salix herbacea through a glacial cycle covering the last 140000 years have shown a decrease in stomatal density in response to the rising CO₂ levels of this period. This accords with the shorter-term observations on leaves of trees seen in herbarium specimens where the stomatal density has decreased in response to the rising CO₂ levels of the last 200 years. The results indicate that natural selection over the 140000-year period may have favoured a similar response to that shown by trees phenotypically over the last 200 years. Since there is now some evidence for the genetic control of stomatal density, the role of natural selection affecting it must be considered when translating responses from short-term experiments to predict how stomatal density will be affected by long-term climatic and atmospheric change.     

Stomatal frequency of Betula pubescens and Pinus sylvestris shows no proportional relationship with atmospheric CO2 concentration

We investigated the relationship between stomatal frequency and a range of atmospheric CO₂ concentrations ([CO₂]_atm) in Betula pubescens and Pinus sylvestris , two important boreal trees in Scandinavia. If strong relationships exist, they can be used to reconstruct past [CO₂]_atm from stomatal frequency of fossil Betula and Pinus leaves. Responses of epidermal characters (stomatal density (SD), epidermal cell density (ED), stomatal index (SI)) to different CO₂ concentrations were investigated utilising (1) the lower partial pressure of CO₂ at increasing altitudes for B. pubescens , and in herbarium specimens of B. pubescens and P. sylvestris collected during the post-industrial rise of [CO₂]_atm from c. 280 ppmv to c. 360 ppmv in 1997 and (2) concentrations (560 ppmv) and temperatures (3° summer) above present day in the CLIMEX greenhouse experiment. All the results show no clear relationship between SD or SI and [CO₂] _atm for either B. pubescens or P. sylvestris. Most likely there are stronger genetically and environmentally induced factors that affect the development of the leaves. Problems with collecting representative samples from herbarium specimens are discussed. Since the effects of changes in [CO₂]_atm cannot be statistically modelled, B. pubescens and P. sylvestris are not suitable for reconstructing past atmospheric CO₂ concentrations from fossil leaves using stomatal density or stomatal index     

Stomatal sensing of the environment

The effects of environmental factors on stomatal behaviour are reviewed and the questions of whether photosynthesis and transpiration eontrol stomata or whether stomata themselves control the rates of these processes is addressed. Light affects stomata directly and indirectly. Light can act directly as an energy source resulting in ATP formation within guard cells via photophosphorylation, or as a stimulus as in the case of the blue light effects which cause guard cell H⁺ extrusion. Light also acts indirectly on stomata by affecting photosynthesis which influences the intercellular leaf CO₂ concentration ( C _i). Carbon dioxide concentrations in contact with the plasma membrane of the guard cell or within the guard cell acts directly on cell processes responsible for stomatal movements. The mechanism by which CO₂ exerts its effect is not fully understood but, at least in part, it is concerned with changing the properties of guard cell plasma membranes which influence ion transport processes. The C _i may remain fairly constant for much of the day for many species which is the result of parallel responses of stomata and photosynthesis to light. Leaf water potential also influences stomatal behaviour. Since leaf water potential is a resultant of water uptake and storage by the plant and transpirational water loss, any factor which affects these processes, such as soil water availability, temperature, atmospheric humidity and air movement, may indirectly affect stomata. Some of these factors, such as temperature and possibly humidity, may affect stomata directly. These direct and indirect effects of environmental factors interact to give a net opening response upon which is superimposed a direct effect of stomatal circadian rhythmic activity.     

Effects of elevated CO2 concentration on photosynthesis, respiration and carbohydrate status of coppice Populus hybrids

To determine how increased atmospheric CO₂ will affect the physiology of coppiced plants, sprouts originating from two hybrid poplar clones ( Populus trichocarpa × P. deltoides - Beaupre and P. deltoides × P. nigra - Robusta) were grown in open-top chambers containing ambient or elevated (ambient + 360 μmol mol⁻¹) CO₂ concentration. The effects of elevated CO₂ concentration on leaf photosynthesis, stomatal conductance, dark respiration, carbohydrate concentration and nitrogen concentration were measured. Furthermore, dark respiration of leaves was partitioned into growth and maintenance components by regressing specific respiration rate vs specific growth rate. Sprouts of both clones exposed to CO₂ enrichment showed no indication of photosynthetic down-regulation. During reciprocal gas exchange measurements, CO₂ enrichment significantly increased photosynthesis of all sprouts by approximately 60% ( P < 0.01) on both an early and late season sampling date, decreased stomatal conductance of all sprouts by 10% ( P < 0.04) on the early sampling date and nonsignificantly decreased dark respiration by an average of 11%. Growth under elevated CO₂ had no consistent effect on foliar sugar concentration but significantly increased foliar starch by 80%. Respiration rate was highly correlated with both specific growth rate and percent nitrogen. Long-term CO₂ enrichment did not significantly affect the maintenance respiration coefficient or the growth respiration coefficient. Carbon dioxide enrichment affected the physiology of the sprouts the same way it affected these plants before they were coppiced.     

Responses of individual stomata in Ipomoea pes-caprae to various CO2 concentrations

The responses of individual stomata to CO₂ concentrations ranging from 0 to 900 μmol mol⁻¹ air were analysed in Ipomoea pes-caprae L. Sweet (Convolvulaceae). The stomata were directly observed using a measurement system that permitted continuous observation of stomatal movement under controlled light and CO₂ conditions. A CO₂ concentration of 350 μmol mol⁻¹ or higher induced stomatal closure, whereas concentrations below 350 μmol mol⁻¹ did not. The time lag before stomatal closure decreased with increasing CO₂ concentration, as did the steady-state aperture of the stomata after a change in CO₂ concentration. However, the rate of stomatal closure increased with increasing CO₂ concentration. Therefore, not only the stomatal closure rate but also the time from the CO₂ concentration change to the beginning of stomatal closure changed with increasing CO₂ concentration. These results suggest that atmospheric CO₂ may be the stimulus for the closure of guard cells. No significant differences were observed between adaxial and abaxial stomata in terms of their responses to CO₂. However, heterogeneous responses were detected between neighbouring stomata on each leaf surface.     

Elevated CO2 concentrations and stomatal density: observations from 17 plant species growing in a CO2 spring in central Italy

Stomatal density (SD) and stomatal conductance ( g _s) can be affected by an increase of atmospheric CO₂ concentration. This study was conducted on 17 species growing in a naturally enriched CO₂ spring and belonging to three plant communities. Stomatal conductance, stomatal density and stomatal index (SI) of plants from the spring, which were assumed to have been exposed for generations to elevated [CO₂], and of plants of the same species collected in a nearby control site, were compared. Stomatal conductance was significantly lower in most of the species collected in the CO₂ spring and this indicated that CO₂ effects on g _s are not of a transitory nature but persist in the long term and through plant generations. Such a decrease was, however, not associated with changes in the anatomy of leaves: SD was unaffected in the majority of species (the decrease was only significant in three out of the 17 species examined), and also SI values did not vary between the two sites with the exception of two species that showed increased SI in plants grown in the CO₂-enriched area. These results did not support the hypothesis that long-term exposure to elevated [CO₂] may cause adaptive modification in stomatal number and in their distribution.     

Modifications of the leaf surface structures of Quercus ilex L. in open, naturally CO2-enriched environments

Two Italian CO₂ springs allowed us to study the long-term effect of a 350–2600 μ mol mol^–1 increase in CO₂ concentrations on the surface structures of leaves of Quercus ilex L. Carbon dioxide increased the quantity of cuticular waxes, above an apparent threshold of 750 μ mol mol^–1 CO₂. Leaf wettability was not modified by CO₂ concentrations. Reduction in stomatal frequency was observable up to 750 μ mol mol^–1 CO₂, the slope being almost the same as that estimated for the increase in CO₂ concentration from pre-industrial times to the present. At higher concentrations, CO₂ seemed to exert no more impact on stomatal frequency.     

Sensing of atmospheric CO2 by plants

Abstract. Despite recent interest in the effects of high CO₂ on plant growth and physiology, very little is known about the mechanisms by which plants sense changes in the concentration of this gas. Because atmospheric CO₂ concentration is relatively constant and because the conductance of the cuticle to CO₂ is low, sensory mechanisms are likely to exist only for intercellular CO₂ concentration. Therefore, responses of plants to changes in atmospheric CO₂ will depend on the effect of these changes on intercellular CO₂ concentration. Although a variety of plant responses to atmospheric CO₂ concentration have been reported, most of these can be attributed to the effects of intercellular CO₂ on photosynthesis or stomatal conductance. Short-term and long-term effects of CO₂ on photosynthesis and stomatal conductance are discussed as sensory mechanisms for responses of plants to atmospheric CO₂. Available data suggest that plants do not fully realize the potential increases in productivity associated with increased atmospheric CO₂. This may be because of genetic and environmental limitations to productivity or because plant responses to CO₂ have evolved to cope with variations in intercellular CO₂ caused by factors other than changes in atmospheric CO₂.     

Photosynthesis, growth and nutrient changes in non-nodulated Phaseolus vulgaris grown under atmospheric and elevated carbon dioxide conditions

The response of Phaseolus vulgaris L. cv. Contender grown under controlled environment at either ambient or elevated (360 and 700 μmol mol^-1, respectively) CO₂ concentrations ([CO₂]), was monitored from 10 days after germination (DAG) until the onset of senescence. Elevated CO₂ had a pronounced effect on total plant height (TPH), leaf area (LA), leaf dry weight (LD), total plant biomass (TB) accumulation and specific leaf area (SLA). All of these were significantly increased under elevated carbon dioxide with the exception of SLA which was significantly reduced. Other than high initial growth rates in CO₂-enriched plants, relative growth rates remained relatively unchanged throughout the growth period. While the trends in growth parameters were clearly different between [CO₂], some physiological processes were largely transient, in particular, net assimilation rate (NAR) and foliar nutrient concentrations of N, Mg and Cu. CO₂ enrichment significantly increased NAR, but from 20 DAG, a steady decline to almost similar levels to those measured in plants grown under ambient CO₂ occurred. A similar trend was observed for leaf N content where the loss of leaf nitrogen in CO₂-enriched plants after 20 DAG, was significantly greater than that observed for ambient-CO₂ plants. Under enhanced CO₂, the foliar concentrations of K and Mn were increased significantly whilst P, Ca, Fe and Zn were reduced significantly. Changes in Mg and Cu concentrations were insignificant. In addition. high CO₂ grown plants exhibited a pronounced leaf discoloration or chlorosis, coupled with a significant reduction in leaf longevity.